Since the outbreak of AIDS and other viral-related disease states there has been an increased need for new and better methods of studying the etiology and pathology of viruses, as well as therapeutic monitoring of treatment regimes for arresting or modifying in some way the effect these viruses have on cells, and ultimately the patient. For example, it is often desirable to assay the percentage of cells infected. A high virus burden usually means the disease is rampant, while a low virus burden might mean that the particular disease is in its early stages, or is responding to therapeutic treatment, and so on. Conventional assays for measuring virus burden have to date been merely extrapolative in nature. For example, cells may be cultured and titrated out for analysis. In these assays, it is common to employ polymerase chain reaction amplification techniques, in an attempt to quantitate copies of DNA (as the provirus form) or RNA present, the precept being, the more RNA, the more virus. However, results obtained with techniques of this sort can only indicate without distinction, a small quantity of infected cells having a high quantity of virus burden, or a larger quantity of cells with a low virus burden per cell. Another drawback is that a determination in accordance with this technique does not provide information as to whether or not the virus infection is replicative or abortive, since one does not know "how many" or "which" cells are infected. Hence, true virus burden assessment, as well as virus activity cannot be had with this technique. Furthermore, it is difficult, cumbersome, and costly to implement, all without giving results that are acceptable in sensitivity.
Virus infection may also be monitored by monitoring the quantity of a viral component such as p24 (in the case of HIV-1) present in a patient's serum. However, this technique is grossly extrapolative and often given to false negatives. For example, a patient may demonstrate a short spike in p24 concentration at the beginning of infection, when the virus is replicating, but before that patient's antibody response to this virus. Within a period of 5 days to about 2-3 weeks, the patient will start to make antibodies to p24. These antibodies bind to the p24 in plasma and either remove it from circulation or block its detection in immunoassays. Accordingly, there is a very short window in which to detect the p24 antigen component, as the patient will test negative once he is making antibodies to the p24. It is not until the patient becomes so compromised that he can no longer produce antibodies to the p24 component, that the test begins to again indicate a positive result for the p24 antigen. Unfortunately, the patient prognosis is very grim at this point, as the disease has progressed past the stage of responding to any therapeutic treatment.
Hence, there is a continuing need for better tools and techniques for measuring viral burden in cells, and the viability and replicability of the virus in those cells. This information is invaluable in determining the progress of the viral infection and its response to treatment. Further, there is a definite need to monitor these parameters at a very early stage in the disease progression, and to continue this monitoring unimpeded throughout the path of infection.
In particular, there is a specific need for routine monitoring of virus load in HIV-infected individuals, preferably, through the use of a fixative that inactivates the virus and thus, increases the safety of handling samples containing this deadly virus. This information will be used by physicians to categorize HIV disease states, monitor and document progression, assess prognosis, and possibly to better tailor effective therapeutic regiments on an individual basis. Additionally, pharmaceutical companies and researchers require just such an assay for use in clinical trials, to determine rapidly if a proposed therapeutic agent is both safe and effective at controlling virus load.
By way of background in cell analysis techniques useful in monitoring a patient, it is noted that conventional flow cytometry is a technique quite suitable for such a task. The fundamental concept of flow cytometry is that cells or subcellular components in aqueous suspension are made to flow at high speed through a sensing region where optical or electrical signals indicative of important biologic properties are generated. These signals are analyzed and accumulated for evaluation. The cells may be fluorescently stained, although other dye systems may be employed, (see U.S. Pat. No. 4,933,293) and no staining is necessary for light-scatter measurements or electrical sizing. Generally, hydrodynamic methods are used to force the cells to move in almost identical trajectories at uniform speeds through a focal spot of intense illumination capable of exciting fluorescent emission from the fluorochrome used. A laser is the typical light source. The cell receives uniform illumination for a very short period of time and emits a burst of fluorescence and scattered light of this duration over all angles. A fraction of the light emission per cell is captured by an optical arrangement and one or more photosensors generating electrical signals proportional to the optical signals. Since the fluorescent light emission occurs at longer wavelength than the incident light while the scattered light experiences no wavelength change, these two signals can be separated with filters and measured independently and simultaneously for each cell. The electrical pulses are shaped, amplified, measured, and either displayed or stored for later analysis. Flow sorting can also be accomplished, to sort different cellular populations from a sample. Typically, the cell suspension is forced out of a tiny orifice and forms a high-speed liquid jet in air. Optical sensing is usually done in the jet in air close to the orifice outlet, and is basically identical to the method used in the flow cytometer just described. Applied ultrasonic vibration causes breakup of the jet into uniform droplets, which traverse a region of high (and constant) electric field intensity. Decision-making and charging circuits electrically charge only droplets containing selected cells; droplets containing unwanted cells and empty droplets remain uncharged. The electric field deflects the charged droplets, which contain the desired cellular subpopulation, into one container while all the other droplets go to another container. In this way specific subpopulations of high purity can be obtained for further biologic study, such as morphologic or biochemical analysis (The above paragraph is taken from: Flow cytometry and Sorting, Melamed et al. editors, John Wiley and Sons, 1979, pp.11-14).
The above-described technique may be used with a patient's cells, to analyze for the presence of viral antigens. Interfering antibodies present in such patient's blood sample are simply washed away with the serum prior to this flow cytometric analysis. However, virion particles, if present, are in the cellular cytoplasm, and sometimes the nucleus. In order to look at the viral antigens inside the cell, the cellular membrane must be permeated to allow antibodies against the virus to enter the cell. Using the prior art techniques of the past, all or a portion of the cellular surface would be stripped away to allow the large antibody to enter. Typically this is done through the use of agents such as methanol or other alcohols which tend to extract lipids and precipitate proteins. Such agents basically turn the cell into a bead of protein, and in this manner provide access to the proteins that may be present. However, in so doing, the cell's surface characteristics are destroyed.
The present invention provides a cellular fixative and fixing technique which fixes cells without substantially destroying that cell's cellular surface characteristics, all the while allowing large molecules, such as antibodies, to enter the cell. This is accomplished without the concomitant release of the virion particles from the inner cell. Thus, a single treatment reagent is provided herein, which is capable of permeating the cell and fixing it, while preserving both the immunoreactivity and light scatter of such cell. The fixative and method of cellular analysis described herein is particularly well-suited for use in flow cytometry analysis.